The development of a Multiplexed Point-of-Care Diagnostic System (MPDS) for rapid diagnosis of patients exposed to biological threat agents is proposed. The system will perform multiplexed measurements to allow for simultaneous detection and identification of multiple agents in both liquid (e.g., blood) and swab samples. Additional capabilities will include multiplexed serological testing of blood for antibody responses to threat agents. The proposed MPDS will consist of two elements: low cost, disposable cartridges and a small portable instrument for reading of cartridges. The disposable, single-use cartridges will contain all reagents necessary to conduct the assays and will use electrochemiluminescent detection. Two cartridge designs are proposed: one to measure analytes in liquid samples, including whole blood and plasma, and another to extract and measure analytes collected on swabs. The cartridges will be automatically processed and analyzed on a compact, portable instrument, providing results in less than 15 minutes. Specific goals of the program include: (1) refinement of MSD's cartridge and cartridge reader designs and demonstration of pilot scale production of both cartridges and reader; (2) development of multiplexed serology and direct detection assays for 10 biothreat agents; (3) transfer of the assay panels to the pilot cartridges and reader; (4) optimization of the assays on the cartridge platform in real matrices; and (5) extensive evaluation of assay performance. On successful completion of the proposed program, the system and assays will be developed to a level where they can subsequently be entered into pre-clinical and clinical validation studies. The proposed test panel for both antigen and serological tests include the following ten threat agents: Bacillus anthracis (BA), Yersinia pestis (YP), Francisella tularensis (FT), Coxiella burnetii (Q Fever), Brucellosis ssp, vaccinia virus, Venezuelan Equine Encephalitis (VEE), botulinum toxin A, ricin, and Staphylococcal enterotoxin B (SEB). In addition to these targets, the panel developed for swab samples will include assays for influenza A, influenza B, and RSV to identify patients whose symptoms are due to common respiratory ailments. Relevance: There is an urgent need for diagnostic systems capable of identifying biological threat agents and the diseases they cause. The proposed diagnostic system will fill that gap by providing rapid point-of-care diagnostics with the reliability and performance of the centralized laboratory. The system will be able to quickly identify individuals exposed to biological threat agents or their toxins, differentiate those individuals infected with common non-threat upper respiratory disease, and allow retrospective serological diagnosis to assist in epidemiological studies. The system is designed to be used by personnel without specialized training and will require no pre-processing of samples. [unreadable] [unreadable]